Method to Improve Lactococcus Preservation

ABSTRACT

Lactococcus lactis  strains with improved preservation characteristics, and improved acid and bile salt tolerance are disclosed. More specifically, a  L. lactis  strain having a heterologous trehalose-6-phosphate synthase gene and/or a trehalose-6-phosphate phosphatase gene, results in an accumulation of trehalose in the cytoplasm and/or in the cytoplasmic membrane. A  L. lactis  strain, having an internal trehalose concentration of at least 10 mg per gram cells (w/w) is disclosed.

The present invention relates to Lactococcus lactis strains withimproved preservation characteristics, and improved acid and bile salttolerance. More specifically, the invention relates to a L. lactisstrain comprising a heterologous trehalose-6-phosphate synthase geneand/or a trehalose-6-phosphate phosphatase gene, resulting in anaccumulation of trehalose in the cytoplasm and/or in the cytoplasmicmembrane. It further relates to a L. lactis strain, whereby saidtrehalose accumulation results in an internal trehalose concentration ofat least 10 mg per gram cells (ww).

Lactococcus lactis is a mesophilic and microaerophilic fermenting lacticacid bacterium. It is commonly occurring in nature, especially on plantmaterial. The bacterium is extensively used in food fermentations,especially in the dairy industry. Moreover, there is an increasinginterest for its use in nutraceuticals, as medication to treat vaginalinfections or as carrier for the delivery of biological activemolecules. In all those cases, there is a need for highly viable startercultures, or pharmaceutical formulations comprising a high proportion ofviable bacteria. One of the major drawbacks of L. lactis however is arapid drop in viability during storage, or during processing for tabletformation. The drop in viability is even more dramatic when thebacterium after lyophilisation is submitted to additional stress such ashigh acidity or the presence of bile salts.

Several methods have been proposed to overcome this problem. Attempts toimprove the viability are made both at the level of culture conditionsof the bacteria as well as at the level of the processing. Gaudu et al.(2002) disclose that Lactococci grown via respiration survive markedlybetter after long time storage than fermenting cells. This long timesurvival is probably due to the induction of cytochromes which mayprotect the cells from oxidative stress. Li et al. (2003) demonstratedthat the presence of intracellular glutathion, which is also protectingagainst oxidative stress, can also result in an improved viability uponstorage.

Another approach to improve the viability of Lactococci upon storagelays in the adaptation of the spray-drying process, and in the use ofprocess aids, such as microcristalline cellulose,carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate,or sodium alginate, which may be used to coat the bacterial particles.

Although these processes certainly lead to an improvement of thestorage, none of the solutions is sufficient, and there is a furtherneed of methods that can lead to an improved storage of Lactococcus,especially in those cases where the bacterium is used for the deliveryof biological active compounds in medical applications.

Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranosyde) is a non-reducingdisaccharide that occurs in a large variety of organisms, ranging frombacteria to invertebrate animals. Trehalose, sometimes in combinationwith dextran is often used as and externally added cryopreservant.Externally added trehalose functions as a saccharide matrix (Conrad etal., 2000), and exerts it protective effect especially during freezedrying, where it acts as a glass former. Moreover, trehalose is wellrecognized as stress metabolite, and it has been extensively studied infungi, especially in Saccharomyces cerevisiae. High concentrations ofinternal trehalose do improve the storage capacity and result in ahigher viability upon cryopreservation. However, it is important to notethat externally added trehalose rarely leads to internal trehaloseaccumulation in micro-organisms, either because it is not taken up, orit is metabolized rapidly after uptake.

Externally added trehalose has been used, amongst others, forpreservation of Lactobacillus during freeze drying (Conrad et al., 2000)and for the stabilization of Lactococcus during freezing (EP 1441027).However, although the role of internal trehalose in eukaryotic cells iswell documented, there are no data available about a positive role inpreservation in prokaryotes.

Padilla et al. (2004) have recently shown that an overproduction oftrehalose can be obtained in the trehalose producing and secretinggram-positive bacterium Corynebacterium glutanicum, by expressing theotsA and otsB genes of Escherichia coli in this species. However, inthis case, the expression of the E. coli genes leads to an increase ofthe endogenous synthase and phosphatase activities, and to an increaseof the existing endogenous trehalose production. Moreover, the effect ofthe overproduction of trehalose on the storage of C. glutanicum isunknown.

Lactococcus lactis is able to utilize trehalose (Andersson et al.,2001), but up to now, no trehalose synthesizing Lactococcus lactisstrain has been described. Indeed, no trehalose-6-phosphate synthase ortrehalose-6-phosphate phosphatase genes has been identified, which areessential steps in the trehalose production starting fromglucose-6-phosphate, a metabolite that is present in L. lactis.Surprisingly we found that, by transfer of and expression in L. lactisof the otsA (trehalose-6-phosphate synthase) and otsB(trehalose-6-phosphate phosphatase) genes of Escherichia coli asignificant trehalose accumulation can be obtained. Even moresurprisingly, this trehalose accumulation leads to an importantimprovement of the viability under stress conditions and during storage,under several storage conditions. Therefore, internal trehaloseaccumulation seems an ideal method to improve the storage and stressresistance characteristics of Lactococcus sp in general and L. lactis inparticular.

A first aspect of the invention is an isolated strain of Lactococcussp., preferably L. lactis, comprising an internal trehaloseconcentration of at least 10 mg trehalose, preferably 30 mg trehalose,more preferably 40 mg trehalose, most preferably 50 mg trehalose pergram wet weight of cells (i.e. 50 mg trehalose, preferably 150 mgtrehalose, more preferably 200 mg trehalose, most preferably 250 mgtrehalose per gram dry weight of cells). Internal trehaloseconcentration as used here means that the trehalose is synthesized ortaken up by the bacteria, and present in the cytoplasm and/or in thecytoplasmic membrane of the bacteria. Internal trehalose differs clearlyform exogenous trehalose, added to the medium, which may stick to theoutside of the bacterial cell wall but is not incorporated in thebacteria. Preferably, said internal trehalose is endogenouslysynthesized trehalose.

A further aspect of the invention is an isolated strain of Lactococcussp, preferably L. lactis, comprising a heterologoustrehalose-6-phosphate synthase and/or trehalose-6-phosphate phosphatasegene. A gene as used here is a DNA sequence that comprises at least thecoding sequences for a functional protein. Preferably, said genes areoperably linked to a promoter that is functional in L. lactis. Operablylinked refers to a juxtaposition wherein the components so described arein a relationship permitting them to function in their intended manner.A promoter sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the promoter sequence. Said promoter may bean inducible promoter or a constitutive promoter. Both genes may beoperably linked to one single promoter, as an operon, or they may beplaced under the control of two promoters, which may be identical ordifferent. One preferred embodiment is an isolated strain of Lactococcussp, preferably L. lactis strain, whereby said genes are placed under thecontrol of the nisin inducible promoter of L. lactis. Another preferredembodiment is a L. lactis strain, whereby said genes are placed underthe control of the constitutive P1 promoter. Preferably, saidtrehalose-6-phosphate synthase gene is the otsA gene of E. coli, and thetrehalose-6-phosphate phosphatase gene is the otsB gene of E. coli.Method for transformation of Lactococcus sp are known to the personskilled in the art, and include, but are not limited to electroporation.The heterologous genes may be situated on a self replicating plasmid, ormay be integrated in the bacterial genome.

Another aspect of the invention is the use of internal trehaloseaccumulation in Lactococcus sp to improve the storage characteristics ofLactococcus. Preferably, said Lactococcus sp. is L. lactis. Trehaloseaccumulation as used here means an internal concentration of trehaloseof at least 10 mg trehalose, preferably 30 mg trehalose, more preferably40 mg trehalose, most preferably 50 mg trehalose per gram wet weight ofcells. Preferably, the accumulation of internal trehalose is obtained byexpression of a heterologous trehalose-6-phosphate synthase and/ortrehalose-6-phosphate phosphatase gene, even more preferably byexpression of the otsA and/or the otsB gene of E. coli.

Alternatively, it may be obtained by external addition of trehalose tothe growth medium, and by replacing the trehalose-6-phosphatephosphorylase by a trehalose-6-phosphatase phosphatase gene such asOtsB, and by growth of this strain on trehalose and another carbonsource, preferably maltose. Indeed, growth on trehalose as such doesn'tlead to trehalose accumulation, due to rapid metabolization afteruptake. Moreover, in L. lactis, trehalose is phosphorylated duringuptake to yield trehalose-6-phosphate. However, by allowing the uptakeof trehalose with the concomitant phosphorylation totrehalose-6-phosphate, but blocking the further down stream processingby converting the trehalose-6-phosphate to trehalose, while thetrehalose-6-phosphate phosphorylase activity is inactivated, anaccumulation of internal trehalose will be obtained. Still anotherpossibility for the accumulation of trehalose in L. lactis is thetransformation of the bacterium with a heterologous non-phosphorylatingtrehalose transporter, such as the Sinorhizobium meliloti trehalose ABCtransporter encoded by the thuEFGK operon (Genbank accession numberAF175299; Jensen et al., 2002), preferably combined with theinactivation of the trehalose phosphotransferase, and the growth of thetransformed strain on trehalose and another carbon source, preferablylactose. Improvement of the storage characteristics as used here can beany storage, such as, but not limited too storage in growth medium,freezing, freeze drying or spray drying. Preferably, said storage isfreeze drying. Improvement of storage can be measured by growing the L.lactis strain under conditions resulting in internal trehaloseaccumulation, freeze-drying the strain, and measuring the evolution ofthe viability of the strain for at least 4 weeks during storage at 4°C., at 10% RH.

Still another aspect of the invention is the use of internal trehaloseaccumulation in Lactococcus sp. to improve the stress resistance ofLactococcus sp. Preferably, said Lactococcus sp is L. lactis. Stressresistance as used here can be any kind of stress. As a non limitingexample, it can be stress induced by cold, stress by freezing, stress byspray drying, stress by freeze drying, stress by highly acidic pH (belowpH 3.5, preferably below pH 3.2, even more preferably below pH 3.0),stress by the presence of bile salts, or a combination of thosestresses, either in parallel or successive. Preferably, said stress isstress by highly acidic pH, even more preferably said stress is stressby the presence of bile salts, most preferably said stress is stress byfreeze drying. A special embodiment is the use of internal trehaloseaccumulation in Lactococcus sp. to improve the stress resistance ofLactococcus sp. to freeze drying, followed by acid stress and/orpresence of bile salts. Preferably, said Lactococcus sp. is L. lactis.Those stress conditions mimic the conditions the bacteria will encounterwhen used for delivery in the intestine.

Still another aspect of the invention is the use of an isolated strainof Lactococcus sp, preferably L. lactis strain according to theinvention for the delivery of a prophylactic and/or therapeuticmolecule. Preferably, said use is the use of an isolated strain ofLactococcus sp., preferably L. lactis, for the preparation of amedicament for delivery of a prophylactic and/or therapeutic molecule.Delivery of biological active polypeptides has been described inWO97/14806. The use of a strain, according to the invention has theadvantage that the production of the prophylactic and/or therapeuticmolecule is significantly higher, both when calculated per colonyforming unit (cfu) or per ml culture. A preferred embodiment is the useof an isolated strain of Lactococcus sp., preferably L. lactis, for thepreparation of a medicament for delivery of a prophylactic and/ortherapeutic molecule, whereby said strain additionally carries aself-containing feature, such as the thyA mutation, disclosed inWO02/090551.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Plasmid map of pNZ8048

FIG. 2: Plasmid map of pNZEcTre1 whereby the otsB/otsA operon isoperably linked to the nisin promoter.

FIG. 3: evaluation of the Trehalose-6-phosphate synthase (marked as 53.6kDa) and trehalose-6-phosphate phosphatase (marked as 29.1 kDa) proteinproduction, under induced (+N) and non-induced (−N) conditions.

FIG. 4: Effect of nisin, used for promoter induction, on the growth rateof the transformants and the non-transformed control strain (+N:induced, −N: non-induced).

FIG. 5: average trehalose accumulation after induction of L. lactisNZ9000 [pN-TRE]

FIG. 6: % survival of L. lactis NZ9000 [pNZEcTre1] after 0.5 h of oxgallchallenge. There percentage is calculated as cfu after treatment oninitial cfu.

FIG. 7: % survival of L. lactis NZ9000 [pNZEcTre1] after freeze-dryingand 4 h of oxgall challenge. There percentage is calculated as cfu aftertreatment on initial cfu.

FIG. 8: % survival of L. lactis NZ9000 [pNZEcTre1] after 0.5 h ofgastric juice challenge. There percentage is calculated as cfu aftertreatment on initial cfu.

FIG. 9: production of human IL-10 after 8 hrs at 37° C., after freezedrying of the culture and rehydratation, calculated per ml culture, byL. lactis NZ9000 [pNZEcTre1-hIL10aPxA] induced (+nisin) and non-induced(−nisin), in comparison with the non-trehalose accumulating controlMG1363 [pT1hIL10aPxA].

FIG. 10: production of human IL-10 after 8 hrs at 37° C., after freezedrying of the culture and rehydratation, calculated per cfu, by L.lactis NZ9000 [pNZEcTre1-hIL10aPxA] induced (+nisin) and non-induced(−nisin), in comparison with the non-trehalose accumulating controlMG1363 [pT1hIL10aPxA].

FIG. 11: Plasmid map of pNZEcTre1-hIL10aPxA whereby the otsB/otsA operonis operably linked to the nisin promoter and the hIL-10 secretioncassette is operably linked to the lactococcal P1 promotor.

EXAMPLES Example 1 Cloning of the Trehalose-Biosynthesis Genes fromEscherichia coli onto the Lactococcal Expression Plasmid pNZ8048

The DNA sequences encoding the trehalose-biosynthesis genes inEscherichia coli are retrieved from GenBank (Acc. Nr. X69160) (Kaasen etal. 1994).

Escherichia coli strain DH5α (Woodcock et al., 1989) serves as thesource of the trehalose biosynthesis genes, otsA and otsB, encodingtrehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase,respectively. Genomic DNA is purified from 10⁹ cells with thecommercially available Qiagen DNeasy kit (Qiagen, Hilden, Germany)according to the supplier's protocol.

The DNA sequence encompassing otsB-otsA is PCR-amplified with Vent® DNApolymerase (New England Biolabs, Beverly, Mass., USA) and the followingprimer sequences:

-   forward primer: 5′-GCCCATGGGTGACAGAACCGTTAACCGAAACC-3′, in which GTG    is the initiator codon of the otsB cistron and the CCATGG sequence    is a NcoI restriction site;-   reverse primer: 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′, in which CTA    is the complement of the TAG stop codon of the otsA cistron and the    TCTAGA sequence is a XbaI restriction site.

The amplified 2216 bp DNA fragment is digested with NcoI and XbaI,ligated into the NcoI-XbaI opened pNZ8048 vector (Ruyter et al., 1996)(FIG. 1) and transformed by electroporation into L. lactis strain NZ9000(Wells et al., 1993). Transformants are obtained at 30C on GM17 (videinfra) plates containing 5 μg chloramphenicol per ml. Plasmid DNA isprepared from the transformants using an SDS-alkaline lysis methodadapted for L. lactis; prior to the production of a cleared lysate thecells are pretreated with lysozyme (5 mg/ml) and mutanolysin (100 U/ml).Combined restriction enzyme digest with BglII and XbaI allowsidentification of the desired recombinant plasmid, designated pNZEcTre0.In this intermediate plasmid construction, the otsB-otsA genes arecloned downstream of the P_(nis) promoter, but the otsB gene is notfused in the correct reading to the ATG initiator codon. The sequence inthis region reads: 5′-GGCACTCACCATGGGTGACAGAA-3′, in which ACA encodesthe 2^(nd) amino acid residue of OtsB. Correct fusion of ACA to ATG isobtained following 3 consecutive PCR amplification steps with Vent® DNApolymerase.

Step 1.

-   Forward primer: 5′-GGCACTCACCATGACAGAACCGTTAACC-3′-   Reverse primer: 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′, in which CTA    is the complement of the TAG stop codon of the otsA cistron and the    TCTAGA sequence is a XbaI restriction site.

The amplified 2216 bp DNA fragment encompasses the otsB-otsA codingregion.

Step 2.

-   Forward primer: 5′-GCGTCGACGGCAATAGTTACCCTTATTATCAAG-3′, in which    GTCGAC coincides with the SalI restriction site in pNZEcTre0-   Reverse primer: 5′-GGTTAACGGTTCTGTCATGGTGAGTGCC-3′, in which CAT is    the complement of the initiator codon preceding otsB.

The amplified 1256 bp DNA fragment encompasses the chloramphenicolresistance gene, the P_(nis) promoter and the nisA ribosome-binding siteand ATG initiator codon fused to the coding region of otsB.

Step 3.

The 2216 bp DNA fragment from step 1 and the 1256 bp DNA fragment fromstep 2 are mixed in equimolar amounts and subjected to PCR amplificationwith Vent® DNA polymerase, using 5′-GCGTCGACGGCAATAGTTACCCTTATTATCAAG-3′and 5′-GCTCTAGACTACGCAAGCTTTGGAAAGGTAGC-3′ as forward and reverseprimers, respectively. The amplified 3444 bp DNA fragment is digestedwith SalI and XbaI and ligated to a SalI-XbaI fragment, carrying thereplicon of pNZ8048. Transformants are obtained in NZ9000 and theirplasmids isolated as described above.

A representative plasmid, whose structure can be identified byrestriction enzyme analysis with SalI, XbaI, BglII, NcoI andcombinations thereof, is designated pNZEcTre1 (FIG. 2). Finally, theregion encompassing the P_(nis) promoter, the nisA ribosome binding siteand the junction of the initiator ATG to the otsB coding region issequence-verified.

Example 2 Induction of the Cloned Trehalose-Biosynthesis Operon in L.lactis

The strains L. lactis NZ9000 [pNZEcTre1] and L. lactis NZ9000 are grownas standing cultures at 30° C. overnight in M17 medium (Difco, Detroit,USA) supplemented with 0.5% glucose (=GM17 medium). The cultures arediluted 100-fold in fresh medium and incubated for another 3 hr at 30 C.The cells are collected by centrifugation and resuspended in theoriginal volume of BM9G medium (M9 medium buffered at pH 8.5 andcontaining 0.5% glucose; Schotte et al., 2000). Nisin (Aplin & Barrett)is added to a final concentration of 0.4 μg/ml and the cultures arefurther incubated for up to 48 hr. At several time points samples aretaken, the cells collected by centrifugation and lysed by addition oflysozyme (5 mg/ml) and mutanolysin (100 U/ml). SDS-PAGE reveals thenisin-dependent induction (Kuipers et al., 1998) of two additionalprotein bands in L. lactis NZ9000[pNZEcTre1]. Their molecular mass of53.6 kDa and 29.1 kDa agree with the molecular mass of the E. colitrehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphatephosphatase (OtsB), respectively (FIG. 3). They are absent from L.lactis NZ9000, irrespective of the addition of nisin. The growth rate ofinduced NZ9000[pNZEcTre1] is severely impaired as early as 3 hr afterthe addition of nisin (FIG. 4). Strain NZ9000 is not affected in itsgrowth rate in the presence of nisin.

Example 3 Optimized Induction Protocol for Trehalose Biosynthesis in L.lactis

Strain NZ9000[pNZEcTre1] is grown to saturation as standing culture at30° C. in GM17Cm (=GM17 containing 5 μg chloramphenicol per ml) anddiluted 3-fold into fresh medium containing 0.4 μg nisin/ml. Incubationis continued at 30° C. with shaking at 200 rpm for 8 hr. The growth rateof the culture is unaffected by the addition of nisin. Saturation isreached after 3 hr of incubation. Induction of OtsA and OtsB can beclearly identified by SDS-PAGE.

Concentration of trehalose is determined by converting trehalose toglucose with trehalase (courtesy of J. Thevelein, Dept. of MolecularMicrobiology, VIB-K.U. Leuven, Belgium), which is measured by a glucoseassay protocol (Trinder, 1969).

The cells are lysed with lysozyme and mutanolysin by incubation in 0.25M Na₂CO₃ for one hr at 37 C and 20 min at 95 C. Cell debris is removedby centrifugation at 13,200 rpm. To one volume of supernatant, 0.5volume of 1 M HAc and 0.5 volume of a buffer, consisting of 300 mM NaAcand 30 mM CaCl₂ pH 5.5, are added. The mixture is incubated for 2 hr at37 C in the presence of trehalase. Following centrifugation at 13,200rpm, the supernatant is supplemented with Trinder reagent (glucoseoxidase, phenol and 4-aminophenazone; Dialab, Austria) and incubatedwith shaking for 15 min at 30 C, after which the OD₅₀₅ is automaticallyrecorded in a 96-well VersaMax tunable microplate reader (MolecularDevices, USA)

Experimental trehalose concentrations are read from a calibration curve,obtained with pure trehalose (Sigma-Aldrich Corp. St. Louis, USA),showing a linear correlation between the OD₅₀₅ value and trehaloseconcentration up to 10 mM trehalose. The accumulation of trehalose inNZ9000[pNZEcTre1] nisin-induced as described above is shown in FIG. 5.The maximum concentration is reached after 3 hr and coincides with thetime point when the culture reaches saturation.

Example 4 Freeze-Drying of L. lactis Cultures and Storage Conditions

Strain NZ9000[pNZEcTre1] is grown to saturation as standing culture at30° C. in GM17Cm and diluted 3-fold into fresh medium with or withoutnisin (0.4 μg/ml). Incubation is continued at 30° C. with shaking at 200rpm for 3 hr. The cells are collected by centrifugation at 5000 rpm,resuspended in the original volume 10% (w/v) skim milk (Difco, BectonDickinson) and kept on ice till ready for freeze-drying.

All freeze-drying runs were performed in triplicate. A sample containingapproximately 2 g of cells (wet weight) is filled in sterile vials(glass type 1, Gaash Packaging, Mollem, Belgium). The vials are coveredwith a lyophilisation stopper (V9032 FM 257/2 SAF1, Bromobutyl withmagnesium silicate as filler, Helvoet Pharma, Alken, Belgium). The vialsare loaded in the pre-cooled production chamber (−25° C.) of thefreeze-dryer (Leybold GT4, Finn-aqua, Santasalo, Sohlberg, Germany)before freezing to −45° C. over a period of 105 min at 1000 mbar. Theprimary drying (12 hr) is performed at −15° C. and 0.8 to 1 mbar; thesecondary drying (9 hr) at 10° C. and 0.1 to 0.2 mbar. Afterlyophilisation, the vials are closed under vacuum.

The vials are stored at different conditions: (a) 8° C. and 10% relativehumidity (RH), (b) 8° C. and 60% RH, (c) 20° C. and 10% RH.

The water content of the freeze-dried culture is determined using aMettler DL35 Karl Fisher titrator (Mettler-Toledo, Beersel, Belgium).The samples are stirred in the reaction medium for 60 s. Afterwards thewater is titrated with Hydranal® Composite 5 (Riedel-de Haën, Seelze,Germany). The analysis is performed in triplicate.

Example 5 Viability of Freeze-Dried Samples of Induced and Non-InducedCultures, Under Different Storage Conditions

To determine the viability in the freeze-dried powder, 0.1 g powder isdissolved in 1 ml sterile water. Viability of the bacteria is determinedby following the growth in a Bioscreen (Labsystems). To this end serialdilutions of the cultures are made, inoculated 1/100 in fresh GM17C andloaded in triplicate into the wells of the Bioscreen. OD₆₀₀ values areautomatically recorded at given intervals over a 21 hr period. The timenecessary to reach an optical density at 600 nm (OD₆₀₀) half way theminimum and maximum OD₆₀₀ (50% time) is calculated based on theexponential growth phase. This 50% time is plotted against the naturallogarithm of the viability and the equation of the standard curve iscalculated. The viability of a freeze-dried sample is determined basedon the standard curve of the starting culture and expressed as % oftheoretical. The viability values of the samples as determined by thismethod corresponds very well with the results obtained by plating andcolony counting.

Table 1 summarizes the measured viability of induced (trehalosecontaining) or non-induced (trehalose free) NZ9000[pNZEcTre1] cultures,freeze-dried and stored under different conditions of temperature andrelative humidity. Viability is expressed as percentage of the viabilityof the respective culture prior to the freeze-drying step.

Example 6 Internal Trehalose Accumulation Protects L. lactis againstOxgall Challenge

A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1] wasinduced by nisin (0.4 μg/ml) for 3 hrs, at 30° C. and 200 rpm in ashacking water bath. The same culture, without nisin addition, was usedas control. The saturated culture was centrifuged and resuspended insterile double distilled water, with different fysiologicalconcentrations of oxgall. The suspensions were incubated for a total of4 hours, at 37° C., and samples were taken after 0, 0.5, 2 and 4 hoursof incubation. The samples were plated on GM17Cm and the plates wereincubated for 24 hours at 30° C. The results were expressed in colonyforming units (cfu; table 2) or as percentage viable colonies, relativeto the initial amount of cfu's (table 3). A graphical representation ofthe results after 0.5 hours of oxgall challenge is shown in FIG. 6.Trehalose accumulation in the nisin induced cultures was checked, andreached a concentration of 60 mg/g ww. Although nisin induction on itsown results in a reduction of cfu in the initial culture, the survivalin the presence of oxgall is clearly better in case of trehaloseaccumulation.

The results are even more pronounced if the culture is freeze driedbefore applying the oxgall stress. A saturated overnight culture of L.lactis NZ9000 [pNZEcTre1] was induced by nisin for 3 hrs, at 30° C. and200 rpm in a shacking water bath. The same culture, without nisinaddition, was used as control. Both sets of culture was freeze dried,and after freeze drying, the powder was dissolved in sterile doubledistilled water and different fysiological concentrations of oxgall wereadded. The suspensions were incubated for a total of 4 hours, at 37° C.,and samples were taken after 0, 0.5, 2 and 4 hours of incubation. Thesamples were plated on GM17Cm and the plates were incubated for 24 hoursat 30° C. The results were expressed in colony forming units (cfu; table4) or as percentage viable colonies, relative to the initial amount ofcfu's (table 5).

The trehalose accumulation in the nisin induced cultures was 58mg/g wwof cells. Cells with internal trehalose accumulation do maintain theirviability better, both after lyophilisation and upon oxgall challenge(Table 4 and 5). FIG. 7 shows the result after 4 hours of oxgallchallenge.

Example 7 Internal Trehalose Accumulation Protects L. lactis againstHigh Acidity in the Medium

A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1] wasinduced by nisin (0.4 μg/ml) for 3 hrs, at 30° C. and 200 rpm in ashacking water bath. The same culture, without nisin addition, was usedas control. Both sets of culture was freeze dried, and after freezedrying, the powder was dissolved in sterile double distilled water anddifferent concentrations of human gastric juice were added(post-operative, pH 2.95)

The suspensions were incubated for a total of 2 hours, at 37° C., andsamples were taken after 0, 0.5, 1 and 2 hours of incubation. Thesamples were plated on GM17Cm and the plates were incubated for 24 hoursat 30° C. The results were expressed in colony forming units (cfu; table6) or as percentage viable colonies, relative to the initial amount ofcfu's (table 7). A graphic representation of the relative results (% cfuafter the treatment calculated on initial cfu) after 0.5 hour is givenin FIG. 8. Internal trehalose accumulation clearly protects L. lactisagainst the high acidity of the gastric juice

Example 8 Internal Trehalose Insures a Higher Productivity of aProphylactic and/or Therapeutic Molecule after Freeze Drying

Construction of pT1hIL10aPxA

The build up of plasmid pT1hIL10aPxA is analogous to the plasmid,containing murine IL-10 (Schotte et al., 2000) It contains the hIL-10gene fused to the usp45 secretion leader, preceded by the coliphage T7gene 10 ribosome binding site and the P1 promoter. The sequence of theIL-10 gene is a synthetic one in which codon usage was adapted to thepreferred codon usage in L. lactis and in which the proline residue—thefirst amino acid of the mature protein in native human IL-10—wasreplaced by an alanine residue. The plasmid was transformed in L. lactisstrain MG1363, according to Wells et al., 1993.

Construction of pNZEcTre1-hIL10aPxA

The plasmid pNZEcTre1-hIL10aPxA is obtained by PCR amplification withVent® DNA polymerase (NEB) of the hIL-10 expression cassette from theplasmid pT1hIL10aPxA and the following primer sequences:5′-GCACTAGTGAATTCGATTAAGTCATCTTACC-3′ and5′-CGACTAGTTAGTTTCGTATCTTCATTGTCATGTAG-3′, in which ACTAGT is a SpeIrestriction site. The amplified 796 bp DNA fragment is digested withSpeI, ligated into the XbaI opened pNZEcTre1 plasmid and transformed byelectroporation into L. lactis strain NZ9000. Transformants are obtainedas described by Wells et al (1993). The direction of the cloned hIL-10expression cassette is sequence-verified (FIG. 11).

A saturated overnight culture of L. lactis NZ9000 [pNZEcTre1-hIL10aPxA]was induced with nisin (0.4 μg/ml), for 3 hours at 30° C., 200 rpm in anorbital shaker. The same culture without nisin was used as control, aswell as a non-induced culture of L. lactis MG1363 [pT1hIL10aPxA]. Thecultures were freeze dried as described in example 4. After freezedrying, the powder was redissolved in the original volume of 50 mM CO₃²⁻, comprising 0.5% glucose. This solution was incubated at 37° C.Samples were taken after 0, 2, 4 and 6 hours, and the amount of hIL-10was determined by an ELISA test (Maxisorp F96 plates (Nunc) were coatedovernight with rat anti-human IL-10 antibody (BD). The plates wereblocked with 0.1% casein solution for 2 hours. Serial dilutions ofrecombinant hIL-10 standard (BD) and supernatants were loaded on theplates. The bound hIL-10 was detected with biotinylated rat anti-humanIL-10 (BD) and horseradish peroxidase coupled streptavidin (BD). Theplates were developed with TMB substrate (BD). The reaction was stoppedafter 30 minutes with 1 M H₂SO₄. The absorbance was measured at 450 nmwith 595 nm as reference wavelength) as well as the cfu by plating. Theresults of the hIL-10 production in function of the culture volume andthe number of cfu, after 8 hours of incubation are shown in FIGS. 9 and10. The production of the trehalose accumulating strain is alwayshigher, independent of the way of calculating the yield, indicating thatnot only the survival is better, but also the production capacity percfu.

Tables TABLE 1 Percentage survival of NZ9000[pNZEcTre1] followingfreeze-drying Storage condition Storage period Induced Non-induceddirectly after freeze-drying  96% 47%  8 C. - 10% RH 1 week 100% 49% 4weeks 109% 37%  8 C. - 60% RH 1 week 103% 15% 4 weeks  86%  6% 20 C. -10% RH 1 week  67% 19% 4 weeks  46%  8%

TABLE 2 Effect of trehalose accumulation in nisin induced cultures onsurvival in different concentrations of oxgall, expressed as colonyforming units after 0 h after 0.5 h after 2 h after 4 h cfu/ml −nisin+nisin −nisin +nisin −nisin +nisin −nisin +nisin before 2.68 × 10⁹ 1.26× 10⁹ 2.68 × 10⁹ 1.26 × 10⁹ 2.68 × 10⁹ 1.26 × 10⁹ 2.68 × 10⁹ 1.26 × 10⁹A.C. after 3.16 × 10⁹ 1.57 × 10⁹ 3.16 × 10⁹ 1.57 × 10⁹ 3.16 × 10⁹ 1.57 ×10⁹ 3.16 × 10⁹ 1.57 × 10⁹ A.C.   0% 3.16 × 10⁹ 1.57 × 10⁹ 3.44 × 10⁹1.56 × 10⁹ 3.29 × 10⁹ 1.48 × 10⁹ 2.84 × 10⁹ 1.36 × 10⁹ oxgall 0.13% 3.38× 10⁹ 1.61 × 10⁹ 1.12 × 10⁹ 1.07 × 10⁹ 2.87 × 10⁶ 4.10 × 10⁷ 6.00 × 10⁴1.48 × 10⁷ oxgall 0.33% 3.43 × 10⁹ 1.30 × 10⁹ 1.12 × 10⁷ 1.44 × 10⁷ 3.00× 10⁴ 7.00 × 10⁴ <1.00 × 10³  <1.00 × 10³  oxgall 0.67% 3.28 × 10⁹ 1.41× 10⁹ 3.51 × 10⁷ 5.38 × 10⁷ 5.06 × 10⁶ 2.05 × 10⁵ <1.00 × 10³  <1.00 ×10³  oxgall

TABLE 3 Effect of trehalose accumulation in nisin induced cultures onsurvival in different concentrations of oxgall, expressed as percentageof the initial concentration after 0 h after 0.5 h after 2 h after 4 h %−nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisin before A.C. 100%100% 100% 100% 100% 100% 100% 100% after 118% 124% 118% 124% 118% 124%118% 124% A.C.   0% 118% 124% 128% 123% 123% 118% 106% 108% oxgall 0.13%126% 128% 42% 85% 0% 3% 0% 1% oxgall 0.33% 128% 103% 0% 1% 0% 0% 0% 0%oxgall 0.67% 122% 112% 1% 4% 0% 0% 0% 0% oxgall

TABLE 4 Effect of trehalose accumulation in nisin induced cultures onsurvival of freeze dried cultures in different concentrations of oxgall,expressed as colony forming units after 0 h after 0.5 h after 2 h after4 h cfu/ml −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisinbefore lyo 3.33 × 10⁹ 1.33 × 10⁹ 3.33 × 10⁹ 1.33 × 10⁹ 3.33 × 10⁹ 1.33 ×10⁹ 3.33 × 10⁹ 1.33 × 10⁹ after lyo 2.02 × 10⁹ 1.28 × 10⁹ 2.02 × 10⁹1.28 × 10⁹ 2.02 × 10⁹ 1.28 × 10⁹ 2.02 × 10⁹ 1.28 × 10⁹   0% 2.02 × 10⁹1.28 × 10⁹ 2.16 × 10⁹ 1.23 × 10⁹ 1.72 × 10⁹ 1.20 × 10⁹ 1.73 × 10⁹ 1.10 ×10⁹ oxgall 0.13% 1.71 × 10⁹ 1.13 × 10⁹ 2.04 × 10⁹ 1.09 × 10⁹ 1.59 × 10⁹1.21 × 10⁹ 1.65 × 10⁹ 1.07 × 10⁹ oxgall 0.33% 1.93 × 10⁹ 1.11 × 10⁹ 1.54× 10⁹ 1.08 × 10⁹ 1.31 × 10⁹ 1.06 × 10⁹ 9.15 × 10⁸ 9.05 × 10⁸ oxgall0.67% 1.29 × 10⁹ 1.07 × 10⁹ 1.14 × 10⁹ 1.11 × 10⁹ 1.04 × 10⁹ 7.53 × 10⁸8.35 × 10⁸ 8.41 × 10⁸ oxgall

TABLE 5 Effect of trehalose accumulation in nisin induced cultures onsurvival of freeze dried cultures in different concentrations of oxgall,expressed as percentage of the initial concentration after 0 h after 0.5h after 2 h after 4 h % −nisin +nisin −nisin +nisin −nisin +nisin −nisin+nisin before 100% 100% 100% 100% 100% 100% 100% 100% lyo After 61% 96%61% 96% 61% 96% 61% 96% Lyo   0% 61% 96% 65% 92% 52% 90% 52% 83% oxgall0.13% oxgall 51% 85% 61% 82% 48% 91% 50% 80% 0.33% 58% 83% 46% 81% 39%79% 27% 68% oxgall 0.67% 39% 81% 34% 84% 31% 57% 25% 63% oxgall

TABLE 6 Effect of trehalose accumulation in nisin induced cultures onsurvival of freeze dried cultures in different concentrations of gastricjuice, expressed as colony forming units after 0 h after 0.5 h after 1 hafter 2 h cfu/ml −nisin +nisin −nisin +nisin −nisin +nisin −nisin +nisinbefore lyo 2.80 × 10⁹ 1.20 × 10⁹ 2.80 × 10⁹ 1.20 × 10⁹ 2.80 × 10⁹ 1.20 ×10⁹ 2.80 × 10⁹ 1.20 × 10⁹ after lyo 2.05 × 10⁹ 1.17 × 10⁹ 2.05 × 10⁹1.17 × 10⁹ 2.05 × 10⁹ 1.17 × 10⁹ 2.05 × 10⁹ 1.17 × 10⁹  0% gastric juice2.05 × 10⁹ 1.17 × 10⁹ 1.82 × 10⁹ 1.25 × 10⁹ 1.75 × 10⁹ 1.38 × 10⁹ 1.86 ×10⁹ 1.20 × 10⁹ 25% gastric juice 2.06 × 10⁹ 8.54 × 10⁸ 1.55 × 10⁸ 7.14 ×10⁸ 1.03 × 10⁸ 6.77 × 10⁷ 6.07 × 10⁷ 1.58 × 10⁷ 50% gastric juice 1.63 ×10⁹ 8.33 × 10⁸ 1.06 × 10⁸ 5.27 × 10⁸ 5.81 × 10⁷ 3.61 × 10⁷ 3.13 × 10⁷1.29 × 10⁷ 75% gastric juice 2.10 × 10⁹ 9.54 × 10⁸ 1.26 × 10⁷ 2.41 × 10⁷9.00 × 10⁶ 1.37 × 10⁷ 4.81 × 10⁶ 9.18 × 10⁶

TABLE 7 Effect of trehalose accumulation in nisin induced cultures onsurvival of freeze dried cultures in different concentrations of gastricjuice, expressed as percentage of the initial concentration. after 0 hafter 0.5 h after 1 h after 2 h % −nisin +nisin −nisin +nisin −nisin+nisin −nisin +nisin Before lyo 100% 100% 100% 100% 100% 100% 100% 100%After lyo 73% 98% 73% 98% 73% 98% 73% 98%  0% gastric juice 73% 98% 65%104% 62% 115% 67% 101% 25% gastric juice 74% 71% 6% 60% 4% 6% 2% 1% 50%gastric juice 58% 70% 4% 44% 2% 3% 1% 1% 75% gastric juice 75% 80% 0% 2%0% 1% 0% 1%

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1. An isolated Lactococcus sp. strain, comprising an internal trehaloseconcentration of at least 10 mg per gram cells (ww).
 2. An isolatedLactococcus sp. strain, comprising a heterologous trehalose-6-phosphatesynthase and/or trehalose-6-phosphate phosphatase gene.
 3. The isolatedLactococcus sp. strain according to claim 2 wherein one or both genesare operably linked to the inducible L. lactis nisin promoter.
 4. Theisolated Lactococcus sp. strain according to claim 2 wherein one or bothgenes are functionally linked to the constitutive P1 promoter.
 5. Theisolated Lactococcus sp. strain according to claim 2, wherein theheterologous trehalose-6-phosphate synthase gene is the OtsA gene fromEscherichia coli.
 6. The isolated Lactococcus sp. strain according toclaim 2, wherein the heterologous trehalose-6-phosphate phosphatase geneis the OtsB gene from Escherichia coli.
 7. The isolated Lactococcus sp.strain according to claim 1 or 2, wherein said Lactococcus sp isLactococcus lactis.
 8. A method of improving storage conditions and/orimproving stress resistance in Lactococcus sp by internal trehaloseaccumulation comprising expressing a heterologous trehalose-6-phosphatesynthase and/or trehalose-6-phosphate phosphatase gene in theLactococcus sp. to improve its storage characteristics and/or stressresistance.
 9. The method of claim 8, wherein said improved storageconditions comprise resistance to freeze drying.
 10. (canceled)
 11. Themethod of claim 8, wherein said improved stress resistance is againstbile salts, acid conditions freeze drying or a combination thereof. 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. The method according toclaim 8, wherein the heterologous trehalose-6-phosphate synthase gene isthe OtsA gene from Escherichia coli.
 16. The method according to claim 8or 15, wherein the heterologous trehalose-6-phosphate phosphatase geneis the OtsB gene from Escherichia coli.
 17. The method according toclaim 8, wherein said Lactococcus sp is L. Lactis.
 18. A prophylacticand/or therapeutic treatment comprising administering a prophylacticand/or therapeutic amount of a composition comprising the Lactococcussp. of claim 1 or 2 to an individual in need thereof.
 19. The methodaccording to claim 8, wherein one or both genes are operably linked tothe inducible L. lactis nisin promoter.
 20. The method according toclaim 8, wherein one or both genes are functionally linked to theconstitutive P1 promoter.